Hydrogenase Activity Research Articles

A Recombinant 12-His Tagged Pyrococcus furiosus Soluble [NiFe]-Hydrogenase I Overexpressed in Thermococcus kodakarensis KOD1 Facilitates Hydrogen-Powered in vitro NADH Regeneration.

Soluble hydrogenase I (SHI) from the hyperthermophilic archaeon Pyrococcus furiosus is a heterotetrameric [NiFe] hydrogenase that catalyzes the reversible reduction of protons by NADPH into hydrogen gas (H2 ). Here, the authors expressed the four αβγδ subunits of SHI encoded by one gene cluster in another hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, which uses its hydrogenase maturation apparatus without the coexpression of native P. furiosus hydrogenase endopeptidases (maturation proteases). The SHI overexpression of T. kodakarensis resulted in more than 1200-fold enhancement in the hydrogenase activity of the cell lysate compared to that of the host strain with an empty vector. An active, purified 12-His tagged recombinant SHI (rSHI) is obtained by one-step affinity adsorption on nickel-charged resin. Size-exclusion chromatography show that purified rSHI is heterotetrameric and has a molecular mass of 150 kDa. The purified rSHI has a half-life of 70 h at 80 °C. This rSHI is used to design a novel in vitro synthetic enzymatic biosystem to convert pyruvate and H2 gas into lactate in a theoretical yield, whereas rSHI is used for NADPH regeneration; an FMN-containing diaphorase (DI) is used to match NADP-preferred SHI and NAD-preferred lactate dehydrogenase (LDH). This study provides a cost-efficient method to obtain hyperthermostable hydrogenases, which can be used in in vitro synthetic enzymatic biosystems for cofactor regeneration and hydrogen production.

Trace metals supplementation enhanced microbiota and biohythane production by two-stage thermophilic fermentation

The effect of trace metals supplementation into palm oil mill effluent on biohythane production and responsible microbial communities in thermophilic two-stage anaerobic fermentation was investigated. High biohythane yields were linked to Ni/Co/Fe supplementation (10, 6 and 20 mg L−1, respectively) with maximum H2 and CH4 yields of 139 mL H2 gVS−1 and 454 mL CH4 gVS−1, respectively. The Ni/Co/Fe supplementation resulted in higher numbers of Bacillus sp., Clostridium sp. and Thermoanaerobacterium sp. together with increasing hydrogenase expression level leading to increasing hydrogen yields of 90.4%. The numbers of Methanosarcina, Methanomassiliicoccus, and Methanoculleus were enhanced by Ni/Co/Fe addition, accompanied by 21.7% higher methane yields. No correlation between methyl coenzyme-M reductase expression level and methane yields was observed. The Ni/Co/Fe supplementation improved gas production in the two-stage biohythane process via enhancing a number of viable hydrogen-producing bacteria together with hydrogenase activity in H2 stage and enhancing number methanogens in the CH4 stage.

Genetic Engineering of an Artificial Metalloenzyme for Transfer Hydrogenation of a Self-Immolative Substrate in Escherichia coli's Periplasm.

Artificial metalloenzymes (ArMs), which combine an abiotic metal cofactor with a protein scaffold, catalyze various synthetically useful transformations. To complement the natural enzymes' repertoire, effective optimization protocols to improve ArM's performance are required. Here we report on our efforts to optimize the activity of an artificial transfer hydrogenase (ATHase) using Escherichia coli whole cells. For this purpose, we rely on a self-immolative quinolinium substrate which, upon reduction, releases fluorescent umbelliferone, thus allowing efficient screening. Introduction of a loop in the immediate proximity of the Ir-cofactor afforded an ArM with up to 5-fold increase in transfer hydrogenation activity compared to the wild-type ATHase using purified mutants.

Improving hydrogen production using co-cultivation of bacteria with Chlamydomonas reinhardtii microalga

Hydrogen production by microalgae is a promising technology to achieve sustainable and clean energy. Among various photosynthetic microalgae able to produce hydrogen, Chlamydomonas reinhardtii is a model organism widely used to study hydrogen production. Oxygen produced by photosynthesis activity of microalgae has an inhibitory effect on both expression and activity of hydrogenases which are responsible for hydrogen production. Chlamydomonas can reach anoxia and produce hydrogen at low light intensity. Here, the effect of bacteria co-cultivation on hydrogen produced by Chlamydomonas at low light intensity was studied. Results indicated that however co-culturing Escherichia coli, Pseudomonas stutzeri and Pseudomonas putida reduced the growth of Chlamydomonas, it enhanced hydrogen production up to 24%, 46% and 32%, respectively due to higher respiration rate in the bioreactors at low light intensity. Chlamydomonas could grow properly in presence of an unknown bacterial consortium and hydrogen evolution improved up to 56% in these co-cultures.

Costless and huge hydrogen yield by manipulation of iron concentrations in the new bacterial strain Brevibacillus invocatus SAR grown on algal biomass

There is a growing global demand on bio-hydrogen production (BHP) using costless and wastes material. Herein we demonstrate the possibility to produce high yield of hydrogen using a new bacterial strain grown on acidic hydrolyzed cyanobacterial biomass as a costless carbon feedstock under various iron concentrations. We used E. coli DH701 mutant and new strain Brevibacillus invocatus SAR isolated from Assiut city soil samples. The mentioned new strain was identified morphologically, biochemically and by molecular analysis using 16S rDNA sequence. Limitation of iron induced BHP in tested cyanobacteria. Iron concentration (0.045 mM) enhanced hydrogenase activity and cumulative hydrogen evolution in the investigated cultures. B. invocatus yielded 3.3 mol H2/mole glucose and 3.8 mol H2/mole reducing sugar (algal biomass), while the mutant strain yielded 1.78 mol H2/mole glucose and 3.4 mol H2/mole reducing sugar (algal biomass). Thus, the use of algal biomass induced higher potency of BHP especially at 0.045 mM iron.

Role of hydrogenases 3 and 4 in Escherichia coli growth and H2 producing hydrogenase activity during anaerobic utilization of lactose

It is known that Escherichia coli produces hydrogen gas (H2) during mixed acid fermentation via hydrogenase (Hyd) enzymes utilizing sugars, alcohols or organic acids. However, there are many sugars including lactose which are not studied well. Therefore, H2 production and responsible Hyd enzymes activity and role have been investigated. During lactose fermentation (1–5 g L−1) by E. coli mainly Hyd-4 and partially Hyd-3 have been shown to affect cell growth and H2 production at different pHs. At 2 g L−1 lactose fermentation in hyfG (lacking large subunit of Hyd-4) mutant at pH 5.5 and 6.5 resulted in the decrease of specific growth rate ∼2.5-fold and ∼1.8-fold, respectively, compared to wild type. Hyd-3 contribution to cell growth was observed at pH 6.5 with 1 g L−1 and at pH 7.5 with 5 g L−1 lactose fermentation. In addition, external pH during 24 or 48 h was less acidified in hyfG mutant cells grown at pH 6.5 than in wild type. Moreover, the H2 yield in wild type cells was 6.00 ± 0.017 mmol L−1. Hyd-3 and Hyd-4 were responsible for H2 production at pH 7.5, 6.5 and 5.5, as shown by using hycE (lacking large subunit of Hyd-3) and hyfG single and hycE hyfB-R (lacking Hyd-3 large subunit and several subunits of Hyd-4) double mutants. These findings provide novel insights into the H2 producing activity of E. coli Hyd-3 and Hyd-4 during utilization of lactose and might be useful for improving H2 production from lactose or lactose containing various wastes.

Hydrogen-Cycling during Solventogenesis in Clostridium acetobutylicum American Type Culture Collection (ATCC) 824 Requires the [NiFe]-Hydrogenase for Energy Conservation

Clostridium acetobutylicum has traditionally been used for production of acetone, butanol, and ethanol (ABE). Butanol is a commodity chemical due in part to its suitability as a biofuel; however, the current yield of this product from biological systems is not economically feasible as an alternative fuel source. Understanding solvent phase physiology, solvent tolerance, and their genetic underpinning is key for future strain optimization of the bacterium. This study shows the importance of a [NiFe]-hydrogenase in solvent phase physiology. C. acetobutylicum genes ca_c0810 and ca_c0811, annotated as a HypF and HypD maturation factor, were found to be required for [NiFe]-hydrogenase activity. They were shown to be part of a polycistronic operon with other hyp genes. Hydrogenase activity assays of the ΔhypF/hypD mutant showed an almost complete inactivation of the [NiFe]-hydrogenase. Metabolic studies comparing ΔhypF/hypD and wild type (WT) strains in planktonic and sessile conditions indicated the hydrogenase was important for solvent phase metabolism. For the mutant, reabsorption of acetate and butyrate was inhibited during solventogenesis in planktonic cultures, and less ABE was produced. During sessile growth, the ΔhypF/hypD mutant had higher initial acetone: butanol ratios, which is consistent with the inability to obtain reduced cofactors via H2 uptake. In sessile conditions, the ΔhypF/hypD mutant was inhibited in early solventogenesis, but it appeared to remodel its metabolism and produced mainly butanol in late solventogenesis without the uptake of acids. Energy filtered transmission electron microscopy (EFTEM) mapped Pd(II) reduction via [NiFe]-hydrogenase induced H2 oxidation at the extracelluar side of the membrane on WT cells. A decrease of Pd(0) deposits on ΔhypF/hypD comparatively to WT indicates that the [NiFe]-hydrogenase contributed to the Pd(II) reduction. Calculations of reaction potentials during acidogenesis and solventogenesis predict the [NiFe]-hydrogenase can couple NAD+ reduction with membrane transport of electrons. Extracellular oxidation of H2 combined with the potential for electron transport across the membrane indicate that the [NiFe}-hydrogenase contributes to proton motive force maintenance via hydrogen cycling.

Enhanced hydrogen production through co-cultivation of Chlamydomonas reinhardtii CC-503 and a facultative autotrophic sulfide-oxidizing bacterium under sulfurated conditions

Photoproduction of H2 using microalgae has been considered as a promising approach for developing sustainable hydrogen energy. The algae C. reinhardtii CC-503 was co-cultured with a facultative autotroph sulfur-oxidizing bacterium Thuomonas intermedia BCRC 17547 to improve H2 production. The maximum H2 production of co-culture at sulfur deficiency conditions was 122 μmol/mg Chl with algae/bacteria ratio as 60:1, which was 2.8-fold higher than that of the pure algal culture. Na2S2O3 treatment can result in a maximum H2 photoproduction rate of 255 μmol/mg Chl, which was 5.9 and 2.1 times higher than those of pure algae culture and co-culture without Na2S2O3. Co-cultivation under sulphate condition can also significantly increase the biomass, respiratory rate, starch content and hydrogenase activity of C. reinhardtii. By supplement of Na2S2O3, persistent (52 days) H2 production of bacteria/algae co-culture can be achieved. Our results demonstrated that co-culture of C. reinhardtii CC-503 and bacteria BCRC17547 is a cost-effective strategy for improving photobiological H2 production.

Helicobacter pylori nickel storage proteins: recognition and modulation of diverse metabolic targets.

Nickel metabolism and trafficking in Helicobacter pylori is complex, perhaps more so than in any other pathogen. Along with nickel enzymes and their associated nickel-binding maturation machinery, H. pylori contains nickel storage proteins, Hpn and Hpnl. Through a combined crosslinking and enrichment approach, we show that Hpn/Hpnl interact with a wide array of partners; over 100 proteins were captured, including known nickel-enzyme maturation proteins, and other proteins outside known H. pylori nickel-associated proteins. The crosslinker binds to exposed amines, but there was no correlation between lysine content and the pulldown abundance of captured proteins. Phenotypic characterization of mutant strains (Δhpn, Δhpnl, or ΔhpnΔhpnl) was used to explore interactions. Nickel deprivation affected the hydrogenase activity of the ΔhpnΔhpnl strain much more severely than the wild-type (WT), whereas the activities of the single mutants were similar to WT. Leucyl aminopeptidase activity was affected in opposite ways in the mutant strains: Δhpn had a threefold decrease, while Δhpnl had a sevenfold increase, compared to the parent. Similar mutant strain analysis supported Hpn and Hpnl acting synergistically to suppress aliphatic amidase activity in a nickel-dependent manner. Recombinant amidase could bind a variety of divalent metals. Amidase activity was greatest in the mutant strains and was inhibited by exogenous nickel. The addition of pure storage protein to extracts from the mutants only restored the suppression of amidase activity for the mutant strain lacking that protein; both storage proteins are needed for amidase suppression. These results suggest that Hpn and Hpnl play more diverse roles than previously thought.

Overproduction of the cyanobacterial hydrogenase and selection of a mutant thriving on urea, as a possible step towards the future production of hydrogen coupled with water treatment.

Using a combination of various types of genetic manipulations (promoter replacement and gene cloning in replicating plasmid expression vector), we have overproduced the complex hydrogenase enzyme in the model cyanobacterium Synechocystis PCC6803. This new strain overproduces all twelve following proteins: HoxEFUYH (hydrogen production), HoxW (maturation of the HoxH subunit of hydrogenase) and HypABCDEF (assembly of the [NiFe] redox center of HoxHY hydrogenase). This strain when grown in the presence of a suitable quantities of nickel and iron used here exhibits a strong (25-fold) increase in hydrogenase activity, as compared to the WT strain growing in the standard medium. Hence, this strain can be very useful for future analyses of the cyanobacterial [NiFe] hydrogenase to determine its structure and, in turn, improve its tolerance to oxygen with the future goal of increasing hydrogen production. We also report the counterintuitive notion that lowering the activity of the Synechocystis urease can increase the photoproduction of biomass from urea-polluted waters, without decreasing hydrogenase activity. Such cyanobacterial factories with high hydrogenase activity and a healthy growth on urea constitute an important step towards the future development of an economical industrial processes coupling H2 production from solar energy and CO2, with wastewater treatment (urea depollution).

Edwardsiella tarda Sip2: A Serum-Induced Protein That Is Essential to Serum Survival, Acid Resistance, Intracellular Replication, and Host Infection.

Edwardsiella tarda is a broad-host pathogen that can infect mammals, reptiles, and fish. E. tarda exhibits a remarkable ability to survive in host serum and replicate in host phagocytes, but the underlining mechanism is unclear. In this study, in order to identify E. tarda proteins involved in serum resistance, iTRAQ proteomic analysis was performed to examine the whole-cell protein profiles of TX01, a pathogenic E. tarda isolate, in response to serum treatment. Of the differentially expressed proteins identified, one (named Sip2) possesses a conserved hydrogenase domain and is homologous to the putative hydrogenase accessory protein HypB. When Sip2 was expressed in Escherichia coli, it significantly enhanced the survival of the host cells in serum. Compared to TX01, the sip2 knockout, TX01Δsip2, was dramatically reduced in the ability of hydrogenase activity, serum resistance, intracellular replication, dissemination in fish tissues, and causing mortality in infected fish. The lost virulence capacities of TX01Δsip2 were restored by complementation with the sip2 gene. Furthermore, TX01Δsip2 was significantly reduced in the capacity to grow under low pHs and iron-depleted conditions, and was unable to maintain its internal pH in acidic environment. Taken together, these results indicate that Sip2 is a novel serum-induced protein that is essential to serum resistance, cellular and tissue infection, and coping with acidic stress via its ability to modulate intracellular pH.

Green Algal Hydrogenase Activity Is Outcompeted by Carbon Fixation before Inactivation by Oxygen Takes Place.

Photoproduction of hydrogen by green algae is considered a transitory release valve of excess reducing power and a potential carbon-free source of sustainable energy. It is generally accepted that the transitory production of hydrogen is governed by fast inactivation of hydrogenase by oxygen. However, our data suggest that photosynthetic electron loss to competing processes, mainly carbon fixation, stops hydrogen production, supports hydrogen uptake, and precedes the inevitable inactivation by oxygen. Here, we show that when transitioning from dark anaerobiosis to light, hydrogen production ceases within 2 min, regardless of the presence of oxygen. Simultaneous monitoring of the active hydrogenase pool size shows that it remains entirely intact up to 4 min after illumination and is inactivated only later. Thus, our data reveal a window of 4 min in which the hydrogenase pool is not being degraded by oxygen. Furthermore, we show that electron loss, prominently to carbon fixation, outcompetes hydrogen production and leads to hydrogen uptake. Indeed, supplying additional reducing power to hydrogenase at the cessation point regenerates the accumulation of hydrogen. Our results imply the fast cessation of hydrogen production is governed by electron loss rather than oxygen inactivation, which takes place minutes later.

Ferric oxide/carbon nanoparticles enhanced bio-hydrogen production from glucose

Hydrogen (H2) gas represents renewable resource candidate to traditional energy resource. Methods to promote the H2 evolution rate attract increasing attentions. In the present research, ferric oxide/carbon nanoparticles (FOCNPs) were synthesized and evaluated the feasibility to enhance the production rate of H2 gas from glucose through anaerobic fermentation by mixed bacteria. The results demonstrated that appropriate dose of FOCNPs promoted, while excessive concentration of FOCNPs suppressed the production of H2. Addition of 200 mg/L FOCNPs resulted in the highest yield of 218.63 ml H2/g glucose, 33.7% higher than 163 ml H2/g glucose of the control test without addition of FOCNPs. However, 400 mg/L of FOCNPs inhibited the H2 evolution to 154 ml/g glucose, 5.5% lower than the control. It was revealed that the H2 evolution followed the acetic pathways. The FOCNPs had a specific surface area of 27.63 m2/g, and thus addition of FOCNPs could provide more attachment sites for the growth of anaerobes. Moreover, FOCNPs could promote the activity of hydrogenase and electron transfer efficiency, beneficial for the bio-H2 evolution. However, excessive addition of FOCNPs could be toxic to microbes, and further suppressing the H2 production. The present research provided an effective method to promote the evolution rate of H2 gas.

The mechanism of photosystem-II inactivation during sulphur deprivation-induced H2 production in Chlamydomonas reinhardtii.

Sulphur limitation may restrain cell growth and viability. In the green alga Chlamydomonas reinhardtii, sulphur limitation may induce H2 production lasting for several days, which can be exploited as a renewable energy source. Sulphur limitation causes a large number of physiological changes, including the inactivation of photosystemII (PSII), leading to the establishment of hypoxia, essential for the increase in hydrogenase expression and activity. The inactivation of PSII has long been assumed to be caused by the sulphur-limited turnover of its reaction center protein PsbA. Here we reinvestigated this issue in detail and show that: (i) upon transferring Chlamydomonas cells to sulphur-free media, the cellular sulphur content decreases only by about 25%; (ii) as demonstrated by lincomycin treatments, PsbA has a significant turnover, and other photosynthetic subunits, namely RbcL and CP43, are degraded more rapidly than PsbA. On the other hand, sulphur limitation imposes oxidative stress early on, most probably involving the formation of singlet oxygen in PSII, which leads to an increase in the expression of GDP-L-galactose phosphorylase, playing an essential role in ascorbate biosynthesis. When accumulated to the millimolar concentration range, ascorbate may inactivate the oxygen-evolving complex and provide electrons to PSII, albeit at a low rate. In the absence of a functional donor side and sufficient electron transport, PSII reaction centers are inactivated and degraded. We therefore demonstrate that the inactivation of PSII is a complex and multistep process, which may serve to mitigate the damaging effects of sulphur limitation.

Iron-sulfur protein maturation in Helicobacter pylori: identifying a Nfu-type cluster carrier protein and its iron-sulfur protein targets.

Helicobacter pylori is anomalous among non nitrogen-fixing bacteria in containing an incomplete NIF system for Fe-S cluster assembly comprising two essential proteins, NifS (cysteine desulfurase) and NifU (scaffold protein). Although nifU deletion strains cannot be obtained via the conventional gene replacement, a NifU-depleted strain was constructed and shown to be more sensitive to oxidative stress compared to wild-type (WT) strains. The hp1492 gene, encoding a putative Nfu-type Fe-S cluster carrier protein, was disrupted in three different H. pylori strains, indicating that it is not essential. However, Δnfu strains have growth deficiency, are more sensitive to oxidative stress and are unable to colonize mouse stomachs. Moreover, Δnfu strains have lower aconitase activity but higher hydrogenase activity than the WT. Recombinant Nfu was found to bind either one [2Fe-2S] or [4Fe-4S] cluster/dimer, based on analytical, UV-visible absorption/CD and resonance Raman studies. A bacterial two-hybrid system was used to ascertain interactions between Nfu, NifS, NifU and each of 36 putative Fe-S-containing target proteins. Nfu, NifS and NifU were found to interact with 15, 6 and 29 putative Fe-S proteins respectively. The results indicate that Nfu, NifS and NifU play a major role in the biosynthesis and/or delivery of Fe-S clusters in H. pylori.

Engineering an [FeFe]-Hydrogenase: Do Accessory Clusters Influence O2 Resistance and Catalytic Bias?

[FeFe]-hydrogenases, HydAs, are unique biocatalysts for proton reduction to H2. However, they suffer from a number of drawbacks for biotechnological applications: size, number and diversity of metal cofactors, oxygen sensitivity. Here we show that HydA from Megasphaera elsdenii (MeHydA) displays significant resistance to O2. Furthermore, we produced a shorter version of this enzyme (MeH-HydA), lacking the N-terminal domain harboring the accessory FeS clusters. As shown by detailed spectroscopic and biochemical characterization, MeH-HydA displays the following interesting properties. First, a functional active site can be assembled in MeH-HydA in vitro, providing the enzyme with excellent hydrogenase activity. Second, the resistance of MeHydA to O2 is conserved in MeH-HydA. Third, MeH-HydA is more biased toward proton reduction than MeHydA, as the result of the truncation changing the rate limiting steps in catalysis. This work shows that it is possible to engineer HydA to generate an active hydrogenase that combines the resistance of the most resistant HydAs and the simplicity of algal HydAs, containing only the H-cluster.

Absolute quantification of selected photosynthetic electron transfer proteins in Chlamydomonas reinhardtii in the presence and absence of oxygen.

The absolute amount of plastocyanin (PC), ferredoxin-NADP+-oxidoreductase (FNR), hydrogenase (HYDA1), and ferredoxin 5 (FDX5) were quantified in aerobic and anaerobic Chlamydomonas reinhardtii whole cells using purified (recombinant) proteins as internal standards in a mass spectrometric approach. Quantified protein amounts were related to the estimated amount of PSI. The ratios of PC to FNR to HYDA1 to FDX5 in aerobic cells were determined to be 1.4:1.2:0.003:0. In anaerobic cells, the ratios changed to 1.1:1.3:0.019:0.027 (PC:FNR:HYDA1:FDX5). Employing sodium dithionite and methyl viologen as electron donors, the specific activity of hydrogenase in whole cells was calculated to be 382 ± 96.5 μmolH2min-1mg-1. Importantly, these data reveal an about 70-fold lower abundance of HYDA1 compared to FNR. Despite this great disproportion between both proteins, which might further enhance the competition for electrons, the alga is capable of hydrogen production under anaerobic conditions, thus pointing to an efficient channeling mechanism of electrons from FDX1 to the HYDA1.

Water-splitting-based, sustainable and efficient H2 production in green algae as achieved by substrate limitation of the Calvin\u2013Benson\u2013Bassham cycle

BackgroundPhotobiological H2 production has the potential of becoming a carbon-free renewable energy source, because upon the combustion of H2, only water is produced. The [Fe–Fe]-type hydrogenases of green algae are highly active, although extremely O2-sensitive. Sulphur deprivation is a common way to induce H2 production, which, however, relies substantially on organic substrates and imposes a severe stress effect resulting in the degradation of the photosynthetic apparatus.ResultsWe report on the establishment of an alternative H2 production method by green algae that is based on a short anaerobic induction, keeping the Calvin–Benson–Bassham cycle inactive by substrate limitation and preserving hydrogenase activity by applying a simple catalyst to remove the evolved O2. Cultures remain photosynthetically active for several days, with the electrons feeding the hydrogenases mostly derived from water. The amount of H2 produced is higher as compared to the sulphur-deprivation procedure and the process is photoautotrophic.ConclusionOur protocol demonstrates that it is possible to sustainably use algal cells as whole-cell catalysts for H2 production, which enables industrial application of algal biohydrogen production.

Photosynthetic biohydrogen production in a wastewater environment and its potential as renewable energy

Oxygen (O2) is a strong inhibitor of hydrogenase (HydA) activity and expression and altering the sulfur (S) oxidizing transitions in photosystem II (PSII) often allows algal photohydrogen production; however, this may not be practical in a wastewater environment. To counteract natural mechanisms of oxygen evolution in PSII, we utilized acetic acid and butyric acid, which are main volatile fatty acids (VFAs) found in anaerobic bacterial digestion in wastewater treatment, as oxygen regulators for photosynthetic biohydrogen production using Chlorella vulgaris. It was found that a VFA-containing synthetic wastewater promotes oxygen depletion in a photobioreactor (PBR), producing maximum hydrogen yield of 65.4 ± 0.3 μmoL H2 L−1 mM−1 acetate without artificial sulfur or chloride deprivation. Butyric acids showed no significant effect on oxygen depletion and biohydrogen production in the PBR. The measurements of both relative expression level of mRNA and specific activities of reactivate HydA revealed that repetitive algal H2 photo-evolution was possible by HydA synthesis in C. vulgaris followed by complete oxygen depletion controlled by acetic acid levels in the PBR. This emerging understanding of the role of VFAs on oxygen regulation in PSII in natural environments is expected to lead algal-driven bioenergy production technologies to the next level.

Free ammonia enhances dark fermentative hydrogen production from waste activated sludge

Ammonium and/or free ammonia (the unionized form of ammonium) are generally thought to inhibit the activities of microbes involved in anaerobic digestion of waste activated sludge. It was found in this work, however, that the presence of ammonium (NH4+-N) largely enhanced dark fermentative hydrogen production from alkaline pretreated-sludge. With the increase of initial NH4+-N level from 36 to 266 mg/L, the maximal hydrogen production from alkaline (pH 9.5) pretreated-sludge increased from 7.3 to 15.6 mL per gram volatile suspended solids (VSS) under the standard condition. Further increase of NH4+-N to 308 mg/L caused a slight decrease of hydrogen yield (15.0 mL/g VSS). Experimental results demonstrated that free ammonia instead of NH4+-N was the true contributor to the enhancement of hydrogen production. It was found that the presence of free ammonia facilitated the releases of both extracellular and intracellular constituents, which thereby provided more substrates for subsequent hydrogen production. The free ammonia at the tested levels (i.e., 0–444 mg/L) did not affect acetogenesis significantly. Although free ammonia inhibited all other bio-processes, its inhibition to the hydrogen consumption processes (i.e., homoacetogenesis, methanogenesis, and sulfate-reducing process) was much severer than that to the hydrolysis and acidogenesis processes. Further investigations with enzyme analyses showed that free ammonia posed slight impacts on protease, butyrate kinase, acetate kinase, CoA-transferase, and [FeFe] hydrogenase activities but largely suppressed the activities of coenzyme F420, carbon monoxide dehydrogenase, and adenylyl sulfate reductase, which were consistent with the chemical analyses performed above.